59 Selected Reaction Monitoring-Based Absolute Quantification of Developmentally Relevant Proteins in Early Bovine Embryos Reveals Differences Between In Vitro and In Vivo Embryo Culture and Between Different Maternal Metabolic Stages

Abstract

Early embryogenesis is a highly complex developmental process, accompanied by a plethora of changes at the morphological and molecular level. Particularly at the level of proteins, these changes are still poorly characterised and understood. During the first cleavages, the embryo depends mainly on maternal transcripts and proteins that were accumulated and stored during oogenesis until embryonic genome activation (EGA) occurs. In the bovine system, the major EGA takes place at the 8- to 16-cell stage. However, we recently demonstrated by liquid chormatography-tandem mass spectrometry (LC-MS/MS)-based holistic proteome approaches that despite transcriptional and translational silencing, the proteome of the early embryo is highly dynamic (Deutsch et al. 2014; Demant et al. 2015). Based on these findings, we established a targeted LC-MS/MS approach based on multiplexed selected reaction monitoring (mSRM), which facilitates an absolute quantification of 27 proteins relevant in early embryogenesis. Each protein is targeted by 2 independent peptides to facilitate highly reliable quantifications. Nine characteristic developmental stages from germinal vesicle oocyte to hatched blastocyst were analysed (n = 6 per stage), and absolute protein contents are reported as femtomole per embryo, with limits of quantification (LOQ) down to 100 attomoles per embryo. Based on their abundance profiles during maturation, zygote formation, and embryonic development, the 27 proteins could be grouped into 6 SOTA clusters. By principal component analysis (PCA), absolute SRM quantifications of only 9 selected proteins were shown to discriminate between all 9 developmental stages analysed, thus providing molecular fingerprints significant for each developmental stage. We used the 27-plex SRM assay as a powerful readout tool and demonstrated substantial quantitative differences between embryos derived from a well-established in vitro culture system and embryos transferred into the oviduct of living animals for 2 days (in vivo culture). Furthermore, in vivo development of embryos in animals differing in their metabolic stress levels led to significant alterations in the 27-plex SRM profiles.

This work was supported by a grant to GJA from Deutsche Forschungsgemeinschaft DFG FOR1041 ‘Germ Cell Potential’ AR 362/7-1 and European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 312097 - FECUND.